The complete and accurate replication of the genome is a central process in biology. This involves multiple processes as well as complex regulatory mechanisms that allow the temporally coordinated duplication of the chromosomes during each cell cycle. Pol plays a crucial role in DNA replication but in addition also is a major participant in DNA repair processes. A knowledge of the properties of human Pol , its regulation and the integration of its functions by protein-protein interactions with other components of the replisome and with DNA repair machinery is thus central to our understanding of human DNA replication and repair. Far less is known about human Pol than its more extensively studied counterpart in budding yeast, an important point because they differ in subunit composition. Our goals are directed toward dissecting the roles of the p12 and p68 subunits in Pol function. Our research is driven by the hypothesis that p12 directly affects the catalytic functions of the polymerase and exonuclease sites of Pol , while p68 plays a role as a scaffold for protein-protein interactions. In Aim 1 we take advantage of recent advances we have made in the expression of recombinant Pol and its subassemblies to make a rigorous biochemical comparisons of the core enzyme (p125/p50), core + p12, core + p68 and the holoenzyme. In Aim 2 we focus on determining the roles by identifying the sites of p12 that allow it to bind to the p125 and p50 subunits of the core enzyme, and the analysis of their individual effects We also will analyze the subunit interactions of Pol and PCNA. In Aim 3 we focus on the functions of p68, with the view that it acts a scaffold that mediates interactions of Pol with other proteins.. We will investigate several hypotheses regarding the mechanisms by which phosphorylation-dephosphorylation regulates protein-protein interactions of p68, and also its role as a targeting protein that recruits protein phosphatase-1. Our studies could contribute to the understanding of cancer etiology, since it is more than likely that alterations in Pol could contribute to genomic instability.